Estrogen induces a homodimeric membrane glycoprotein (EIMG) with a subunit molecular weight of Mr 104 kDa in the chicken oviducts. It possesses transferrin binding properties similar to transferrin receptors (Tf). However, it is an intracellular protein, structurally distinct from transferrin receptor (TfR) and not involved in transferrin endocytosis. Amino terminal and internal peptide sequence analysis of EIMG showed that eleven out of 14 peptides are highly homologous with human tumor rejection antigen gp96, heat-shock protein hsp108, murine endoplasmic reticulum protein gp99 and hamster glucose regulated protein. All these proteins are homologous to each other and to yeast hsp90. The estrogen-inducible membrane glycoprotein is also induced by stresses such as heat-shock. It appears to be related but distinct from hsp108. It is synthesized as at least two species of molecular masses 104 kDa and 116 kDa. The 116 kDa appears to undergo cleavage by an unidentified protease to generate 104 kDa. Although both species bind transferrin, the 104 kDa species seem to have higher affinity for transferrin. The 104 kDa species, but not the 116 kDa species are expressed in breast cancer cell lines. The current proposal is aimed on elucidating the structure and investigating its functional roles. We propose to investigate whether EIMG is involved in the following functional roles: 1) a chaperone in translocating Tf-TfR from the cell surface into the cell and apoTf-TfR back to the cell surface, 2) protein folding similar to ER localized glucose regulated proteins. The chaperone function in Tf-TfR translocation will be investigated by testing the binding of radiolabelled Tf-TfR complexes to immobilized EIMG. To study whether it is involved in protein folding we will chromatograph the oviduct membrane extract on immobilized denatured proteins. If it is involved in folding, it will bind to denatured but not non-denatured proteins and elute with ATP. To get an insight into its functional roles, we will also study its primary structure. Our strategy is, first we will identify more peptide sequences unique to EIMG by sequencing internal peptides of 104 kDa and 116 kDa and end group analysis of 116 kDa species. We will make use of the unique sequence information to clone the cDNA for EIMG. Our first approach will be to design nested primers and amplify the EIMG specific gene sequences by Rapid Amplification of cDNA (RACE) from isolated RNA. An alternate approach will be by screening the cDNA libraries with degenerate primers synthesized using amino acid sequence information. The derived sequence information will be compared with related chaperone proteins to determine its common functional roles. Our long-term goals are to study the expression of EIMG in normal mammary and breast cancer tissues and correlate with prognosis and association with drug resistance.